Reinoculation and two-dimensional electrophoresis were performed to analyze the pathogenicity differentiation of Curvularia lunata.Resistant host inbred lines Shen135,Mo17,and 78599-1 were reinoculated(six generations) with low virulent isolate WS18.Results showed that the disease index had no significant change for the first 2 generations of inoculation.At the third generation,the incidence of disease was increased and the number of differential expressed proteins of mycelia were more than that in the first 2 generations.More than 100 differential expressed proteins were found in the mycelia of fifth generation when compared with the original one.In the experiment,10 differentially expressed proteins were identified by MALDI-TOF-MS analysis.Three proteins were related directly to the differentiation of virulence,2 were related to allergen,4 were related to the metabolism of carbon or signaling pathway and 1 was unkonwn to Curvularia lunata.
One-dimensional electrophoresis （1-DE） of proteins, two-dimensional electrophoresis （2-DE） of proteins and cloning of cDNA sequence were used to study the virulence differentiation of Curvularia lunata （Wakker） Boed. isolated from maize （Zea maydis L.） in China. From 1-DE gel profiles of proteins, 110 reproducible bands were separated from six isolates of C. lunata CX-3, SD-6, C-152, C107-1, DD-60 and W-18. Sixty-eight bands （61.82%） were polymorphic, suggesting huge biodiversities among the isolates. All isolates for the experiment were clustered into three groups consisting of different virulent types by coefficient value of 0.605. Group 1, consisting of CX-3, SD-6 and C- 152 with high virulence displayed more protein bands than Groups 2 and 3, consisting of C107-1 and DD-60 with low virulence. Proteomics approaches based on 2-DE techniques were applied to identify specific proteins associated with the virulence differentiation in CX-3 and DD-60. A total of 423 protein spots were separated. Out of them 75 specific protein spots were displayed in 2-DE gels. Among them 28 protein spots were unique in CX-3 and eight in DD-60, and 39 protein spots were shown on both 2-DE gels but expressed differently in intensity. Twenty protein spots including three unique protein spots and 17 differentially expressed protein spots （more than two-fold DD- 60） in CX-3 were further identified with MALDI-TOF MS/MS. Results indicated that most of the identified proteins were found to be associated with virulence differentiation, metabolisms, stress response and signal transduction. One of them was identified as Brnl protein, which had been reported to be related to melanin biosynthesis and the virulence differentiation in fungi. Combined with our previous findings, we assumed that Brnl protein and its regulating products might be involved in the virulence differentiation of C. lunata. Consequently, we cloned a Brnl cDNA fragment and aligned it with the fragments in other fungi. Results indicated that the 633-b
Shufa Xu Jie Chen Lixing Liu Xiaofei Wang Xiuli Huang Yuhong Zhai
